Tuberculosis (TB) is a major cause of morbidity and mortality throughout the World. It is estimated that one person develops TB every four seconds and someone will die from the disease every 20-30 seconds. Adding to this, approximately ⅓ of the world's populations is latently infected with Mycobacterium tuberculosis (M. tuberculosis), the causative agent of TB.
Immunocompetent individuals infected with M. tuberculosis in general have a lifetime risk of 10% of developing active TB disease, with this risk increasing dramatically e.g. if the individual is co-infected with HIV (Sonnenberg, 2005) or have diabetes (Young, 2009). If left untreated, each person with active pulmonary TB will infect 10-15 people each year (World Health Organization Tuberculosis Fact Sheet No 104, 2002). Thus, it is important to be able to detect M. tuberculosis-infected individuals at an early stage of infection in order to prevent the progression of M. tuberculosis-infection to active contagious pulmonary TB. This can be achieved by prophylactic treatment at the earliest time point possible after diagnosis. Therefore, a fast and accurate diagnosis of M. tuberculosis infection is an important element of global health measures to control the disease.
Current diagnostic assays to determine M. tuberculosis infection include culture, microscopy and PCR of relevant patient material, chest X-rays, the standard tuberculin skin test (TST), and Interferon-gamma release assays (IGRAs). The three first methods are used for diagnosing active contagious TB and are based on the identification of the M. tuberculosis bacteria and therefore depend on the presence of bacteria in the sample. This demands a certain bacterial load and access to infectious material and the methods are therefore not suitable for early diagnosis of infection i.e. before the onset of clinical disease. Chest X-ray is insensitive and only applicable for pulmonary TB and for detecting TB in a more progressed stage.
The standard tuberculin skin test, displaying a delayed type hypersensitivity reaction (DTH), is a simple and inexpensive assay based on immunological recognition of mycobacterial antigens in exposed individuals. However, it is far from ideal in detecting M. tuberculosis infection. It employs intradermal injection of purified protein derivative (PPD) which is a crude and poorly defined mixture of mycobacterial antigens some of which are shared with proteins from the vaccine sub-strain M. bovis bacille Calmette-Guerin (BCG) and from non-tuberculosis environmental mycobacteria. This broad cross-reactivity of PPD causes a poor specificity of TST, leading to a situation where BCG vaccination and exposure to non-tuberculosis mycobacteria gives a test result similar to that seen in M. tuberculosis-infected individuals.
M. tuberculosis infection mediates a strong cell mediated immune (CMI) response and detection of immune cells and any product deriving from such cells that are generated as a part of the specific response to M. tuberculosis-infection would be a suitable method to detect infection (Andersen, 2000). In order to generate such a specific response, the reagent must 1) be broadly recognized by M. tuberculosis-infected individuals, and 2) be specific for M. tuberculosis thereby discriminating between TB infection, BCG vaccination, and exposure to non-tuberculous environmental mycobacteria.
The genome of M. tuberculosis predicts 4018 open reading frames (tuberculist.epfl.ch/, release R27—March 2013). However, only a minority of these are T-cell antigens with a strong and broad recognition by peripheral blood mononuclear cells (PBMCs) from human TB patients. Algorithms to predict T cell epitopes have been developed, but experimental verification was essential to identify the immunodominant antigens CFP10 and ESAT-6. The M. tuberculosis extracellular or culture filtrate (CF) proteins constitute a protein fraction enriched in T cell antigens (Andersen 1994) and separation of CF proteins by a two-dimensional proteomics based approach led to identification of 59 human T cell antigens of which 35 had been described before (Deenadayalan, 2010). Although this list may not be exhaustive it emphasizes that only a minor part of the approx. 900 CF proteins (Albrethsen, 2013) are T-cell antigens. Logically one would expect that the most immunodominant antigens encoded from the M. tuberculosis genome would be the subset with the highest expression and with current technology it would be possible to rank the genes according to transcription levels under certain growth conditions. However, the contribution that the transcription level makes to the immunogenicity is low and it cannot be used systematically to pinpoint which genes encode relevant antigens (Sidders, 2008).
A highly specific reagent candidate could be sought among antigens from the RD regions (regions of deletion) of the M. tuberculosis genome. These regions represent genomic deletions from the M. bovis BCG vaccine strain compared to the virulent M. tuberculosis strain (Behr, 1999). Therefore, in theory, proteins from these regions (RD proteins) would be excellent candidates as TB diagnostic reagents, i.e. they should not be recognized by healthy un-infected individuals independent of their BCG vaccine status or exposure to non-pathogenic mycobacterial strains. However, of all the predicted genomic ORF's (open reading frames) deleted from BCG it is not known per se which ones are in fact expressed as proteins and furthermore the immunoreactivity remains unknown until tested with sensitized lymphocytes from M. tuberculosis-infected individuals. This can e.g. be done in a whole blood assay, by re-stimulation of PBMCs or by injecting the substances into skin similar as PPD/Mantoux test is administered. E.g. evaluation of Rv3872 showed low interferon gamma (IFN-γ) responses in the tested human TB patients (n=7) 1-4 months after diagnosis (WO 99/24577), demonstrating that this RD protein is not frequently recognized, and Rv3872 was therefore not further pursued for a CMI based diagnostic test.
Potential specific M. tuberculosis proteins are not limited to RD proteins as e.g. the EspC protein (Rv3615c) is specifically recognized in human TB patients and M. bovis infected cattle and not in BCG vaccinated/infected even though the gene is present in BCG (WO2009060184; Sidders, 2008; Millington, 2011). The lack of reaction in BCG-vaccinated individuals is most likely because secretion of Rv3615c is abolished in BCG since it depends on the ESX-1 secretion system which is partly located in the RD1 locus and which is absent in BCG. This antigen is a CMI diagnostic reagent considered as potent as ESAT-6 and CFP10 inducing a comparable strong IFN-γ response (ibid).
When a potentially specific T-cell antigen has been identified, it should be verified that the antigen is specifically recognized in M. tuberculosis infected individuals and not in BCG vaccinated persons. For Rv3873 from RD1 it appeared that the protein was a member of a protein family with a conserved motif in amino acids 118-135 also present in other M. tuberculosis proteins. As a broadly recognized T-cell epitope was present in this motif, BCG vaccinated individuals also responded to the peptide spanning this sequence (Liu, 2004). However, cross-reactivity was also observed to Rv3878 and Rv3879c although no homology was detected by comparing with sequences of other known mycobacterial proteins (Liu, 2004) emphasizing that the specificity of a potential diagnostic candidate needs to be experimentally verified. In the same manner, Rv2653c from RD13 was recognized both in BCG vaccinated donors and TB patients although database searches with the BLAST algorithm did not reveal any obvious mycobacterial protein which could explain the observed cross-reactivity (Aagaard, 2004).
Having identified M. tuberculosis specific proteins with potential for diagnosis of M. tuberculosis infection by CMI based assay it remains to be investigated if a pool of such proteins/peptides will provide the desired sensitivity for a diagnostic test. It is not possible to predict how much a given antigen will add to the sensitivity after combination to already defined diagnostic antigens; this has to be experimentally evaluated for each antigen, and preferably in TB patients from different parts of the World with different genetic backgrounds.
The diagnostic potential of CFP10 (Rv3874), ESAT-6 (Rv 3875), two low molecular proteins from the RD1 region, and TB7.7 (Rv2654) is very well documented (Brock, 2004; Moon, 2013, WO2004099771) and are currently used in different diagnostic reagents registered for human use. Peptides covering CFP10 and ESAT-6 are used in the T-SPOT®.TB test, which is a cellular blood test that detects the immune response of T cells found in PBMCs that have been re-stimulated with ESAT-6 and CFP10. This response is detected by a highly sensitive enzyme-linked immunospot methodology, designated ELISPOT, and is commercialized as the T-SPOT.TB test. This test is highly sensitive and independent of BCG vaccination status. Another registered test for detection of M. tuberculosis-infection is QuantiFERON®-TB Gold, which is an in-vitro diagnostic technology enabling detecting of immune responses in whole blood samples upon re-stimulation with peptides covering ESAT-6, CFP10 and a single peptide from TB7.7. Both of these tests measure the production of interferon-gamma (IFN-γ) in response to exposure with the selected specific antigen peptide pools and are at present considered state-of-the-art. T-SPOT.TB and QuantiFERON®-TB Gold test are collectively recognized as IFN-γ release assays (IGRAs).
Other cytokines and chemokines than IFN-γ have also shown relevance when monitoring the immunological response to mycobacterial antigens. IFN-γ-induced protein (IP-10) is expressed at 100-fold higher levels compared to IFN-γ and diagnostic assays based on the secretion of IP-10 have shown diagnostic performance comparable to IFN-γ release assays (Ruhwald, 2009).
In addition to these in vitro tests, which are already registered for human use and used throughout the world, ESAT-6 and CFP10 have also proven to be effective as skin-test reagents. Clinical studies have shown that a skin test applied in the same way as PPD but using ESAT-6 and CFP10 produced and delivered as recombinant proteins can be used to diagnose M. tuberculosis infection and is un-affected by the BCG vaccination status (Aggerbeck, 2013).
Despite the widespread use of BCG and several diagnostic methods including IGRA, TB keeps taking its toll with almost two million deaths a year and there is a continued need to develop immunodiagnostic tests with improved sensitivity. Immunocompromised patients are at higher risk of developing TB and unfortunately both TST and IGRAs in their present form, performs suboptimal in these groups. The patient groups in highest need of improved testing comprise: HIV-infected patients, patients with immune mediated inflammatory diseases, patients receiving immune suppressive medication (e.g. prednisolone or TNF-α inhibitors) and patients with chronic renal failure. In e.g. HIV infected it is well known that a low CD4 cell count (e.g. <250 cells/pi) is strongly associated with higher rates of indeterminate test outcome, compromised test sensitivity for active TB and decreased likelihood for positive test response in exposed individuals reviewed in (Redelman-Sidi, 2013).
Another very relevant group for targeted testing is children. The diagnosis of latent TB infection (LTBI) and TB in children is difficult, microbiological confirmation of infection is often not obtained and treatment is directed by the clinical presentation alone. In both active and presumed latently infected young children, the immune system is immature, and is the likely cause of lower cytokine release and compromised IGRA performance. Recently it was shown that the QuantiFERON©-TB Gold test had a sensitivity of 53% in 81 children with microbiologically confirmed TB, underpinning the need for improved immunodiagnostic tests for M. tuberculosis infection in children (Schopfer, 2013).
The core problem with the IGRA test performance in the high-risk patient groups mentioned above (e.g. Immunosuppressed, HIV infected, Children) is that the underlying immunosuppressive condition that drives the increased risk of TB disease in itself is characterized by low CMI responses and low IFN-γ release in response to antigens. As the IGRA result is determined based on comparison of the magnitude of IFN-γ release to a cut off, a compromised IFN-γ release increases the risk of the test result becoming false negative. Therefore, it is obvious to the skilled addressee that including more specific antigens will recruit more specific T cells and result in an augmented CMI response and release of IFN-γ and consequently lowering the risk of the response falling below the cut off. Therefore adding additional specific antigens addresses a major limitation in the IGRA tests by improving diagnostic sensitivity.
Another benefit from diagnosing infection of M. tuberculosis based on responses of higher magnitude is an increased analytical accuracy and more reliable test results. In the QuantiFERON®-TB Gold test the cut off for positive test is 0.35 IU/ml or 17.5 pg/ml, a very low concentration which is difficult to determine with high precision—even with sensitive methods as ELISA. For example, the largest precision study of an IGRA to date, found considerable variability in TB response measured by QuantiFERON-TB Gold In-Tube on retesting of the same patient sample. Variability within individuals included differences up to 0.24 IU/ml, in either direction, when the initial response was between 0.25 and 0.80 IU/ml. This led to the conclusion that positive QuantiFERON TB Gold In-Tube test results less than 0.59 IU/ml should be interpreted cautiously (Metcalfe AJRCCM 2012).
Modelling studies suggest that without new vaccines, TB cannot be eliminated and novel and more effective vaccines are an international priority. The overall idea is to supplement the current BCG vaccine with a booster subunit vaccine or creating a novel live TB vaccine to replace BCG. There are an increasing number of experimental vaccines in clinical development and the emerging consensus is that ESAT-6 appears to be an essential vaccine antigen. Thus, many of the novel vaccines currently at the preclinical level or in clinical testing contain ESAT-6. Recently, Aeras Foundation announced the first-in-man trial of an ESAT-6-containing vaccine designed to protect people already latently infected with TB, from developing active TB disease (Aagaard, 2011). Several live vaccine candidates are also directly recombinantly engineered to express ESAT-6 e.g. rBCG:GE (Yang, 2011), rM.S-e6c10 (Zhang, 2010), Salmonella/Ag85B-ESAT-6 (Hall, 2009), rBCG-A(N)-E-A(C) (Xu, 2009) or fusion proteins incorporating ESAT-6 e.g. H1 (van Dissel, 2010; van Dissel, 2011). Unfortunately, the use of ESAT-6 based diagnostics in the IGRA test and vaccination with an ESAT-6 containing vaccine is an exact repetition of the cross-reaction problem associated with the parallel use of TST and BCG.
Consequently, there is a great need for a specific diagnostic reagent that can be used in parallel with both BCG and ESAT-6 containing vaccines. Using in vivo or in vitro assays the reagent should be able to detect M. tuberculosis infections in humans and animals and to discriminate not only between TB infection and vaccination with BCG or the novel ESAT-6 containing vaccines but also exposure to non-pathogenic environmental mycobacteria. The diagnostic reagent should have at least the same sensitivity as the current combination of ESAT-6, CFP10 and in some diagnostic assays TB7.7.
EP2417456 describes such a system where using Rv3615c in conjunction with CFP-10 provides diagnostic sensitivity very similar to the ESAT-6/CFP-10 combination.
Because of the unique characteristics of ESAT-6 being highly immunogenic and specific for M. tuberculosis-infection, it is not likely that replacing ESAT-6 with a single antigen will increase sensitivity compared to ESAT when studying various population groups. This has e.g. be demonstrated by Brock et al. showing recognition of single antigens between 14-43% in TB patients compared to ESAT-6 giving rise to a response in 75% in same patient group. Given that the majority of antigens are less immunogenic compared to ESAT-6, it is more likely that a pool of antigens is needed for responses of high magnitude and improved diagnostic sensitivity.
As exemplified it is not simple to predict the sensitivity and specificity of antigens combinations; rather this requires a detailed design of specific antigen combinations. Adding to this, by increasing the number of peptides in the diagnostic pool it introduces the risk of decreasing the specificity further by increasing the numbers of false positives emphasizing that the diagnostic or immunogenic compositions for specific diagnosis of TB needs to be carefully selected and tested.
There is therefore an urgent need for improved in vivo or in vitro cell-mediated immunological diagnosis of infection with M. tuberculosis in a human or animal. That is a need for antigen combinations that increases the sensitivity (that gives less false negatives) compared to the existing antigen combinations without compromising the specificity (amount of false positives). The needed improved antigen combinations relates to both antigen compositions not including the ESAT-6 antigen to anticipate the situation when a ESAT-6 comprising vaccine is introduced, and antigen combinations comprising ESAT-6 to improve the present state-of-the-art diagnostic reagents.
Our data demonstrate that the CFP10/ESAT6 and the CFP10/Rv3615c combinations can be further improved by adding peptides derived from three novel antigens with diagnostic potential. This novel finding is unexpected for two reasons:                a) The majority (>99%) of the antigens on the TB genome are non-specific and shared among various mycobacterial species so identifying strongly recognized antigens that are specific for Mycobacterium tuberculosis has been very difficult        b) The sensitivity of the CFP10/RV3615c and CFP10/ESAT6 diagnostic combination are already very high so increasing the sensitivity even further becomes increasingly difficult due to non-specific responses.        
The present invention is therefore very encouraging as it describes peptides with the ability not only to increase sensitivity of the CFP10/RV3615c combination but also of the current diagnostic cocktail that includes ESAT6—and without compromising specificity.